Structure of the C-terminal domain of transcription factor IIB from Trypanosoma brucei

Abstract

In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 Å resolution structure of the C-terminal domain of tTFIIB (tTFIIB C ). The tTFIIB C structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIB C contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosomespecific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms.eukaryotic transcription ͉ parasite ͉ RNA polymerase II ͉ trypanosome T rypanosomes are flagellated protozoa, belonging to the order Kinetoplastida, that are exclusively parasitic (1). These organisms reside in insect vectors that transmit the parasites to mammals, birds, fish, and plants. Trypanosoma brucei and Trypanosoma cruzi are of particular medical concern because they cause debilitating and fatal diseases in millions of people annually in tropical regions of the world (2). Trypanosomes diverged from other eukaryotes early in evolution, and thus many biological processes that are well understood in metazoans are highly distinct in these parasites (3, 4). For example, little is known about how trypanosome RNA polymerase II (tRNAP-II) transcription is initiated. It is known that tRNAP-II transcribes most protein-encoding genes, which are arranged in tandem arrays, into polycistronic mRNAs (5). Thus far, the only tRNAP-II promoter that has been identified is the spliced leader (SL) RNA gene promoter (6). The SL RNA gene codes for the SL, which is capped and added in a trans-splicing reaction to the 5Ј end of each ORF contained in a polycistronic mRNA. The addition of an SL to every mRNA is a universal requirement in trypanosomes; therefore, understanding transcription initiation at the SL RNA gene promoter is a crucial step toward understanding the control of tRNAP-II transcription in these parasites (4, 7).To date, components of the SL RNA transcriptional machinery have been studied by using genomics or biochemistry to identify candidate proteins followed by a combination of phenotypic analysis of protein function using either RNAi-silencing of endogenous protein in parasites or depletion/add back studies of in vitro transcription systems. T...

A TFIIH-Associated Mediator Head Is a Basal Factor of Small Nuclear Spliced Leader RNA Gene Transcription in Early-Diverged Trypanosomes

“…Initially, a USE-binding complex was characterized, which consisted of three SNAP c subunits, TRF4, a clear ortholog of the small TFIIA subunit (TFIIA-2), and a sixth protein whose sequence conservation was too weak to identify it unequivocally as the second, larger TFIIA subunit (13,40). Subsequently, a TFIIB ortholog which deviates substantially in sequence and structure from its human counterpart was identified (20,34,39). The identification of TFIIA-2 and TFIIB suggested that trypanosomes harbor a full set of GTFs.…”

TbENF is an essential TbTFIIB-interacting trypanosomatid-specific factor

“…The fact that Tb RNAPII was reported to interact with ENF but is not seen here may reflect interference by the GST-tag with certain protein interactions [39]. Curiously, the crystal structure of Tb TFIIB reveals the trypanosome factor has additional structure relative to TFIIB from higher eukaryotes [13]. It is speculated that these extra regions allow for interactions with trypanosomatid-specific factors.…”

“…For example, the trypanosomatid general transcription factor TFIIB is divergent in its sequence [11, 12] and the crystal structure reveals the presence of extra helices and loops speculated to participate in trypanosome-specific protein interactions [13]. The composition of Tb TFIIH was examined and found to contain essential subunits unique to the parasite, while lacking the cyclin-activating kinase [14, 15].…”

RNA polymerase II transcription: Structure and mechanism

“…The divergent TFIIB from the parasite Trypanosoma brucei lacks an extensive basic surface for promoter binding, and it is also devoid of the structural and sequence features for interaction with BRE u , which may account for the observed bidirectional transcription [38–41]. Widespread divergent transcription has recently been reported for active promoters in other species including mammals and yeast [42, 43].…”